Cancer is a deadly disease causes more 500,000 deaths annually in the United States. Although huge effects have been made, the cancer therapy is restricted by still lack of effective and reliable methods for early diagnostics and evaluation of tumor biomarkers. Therefore, it is critical to develop an effective way to detect the tumor and its biomarkers in the early stage.
Multiple approaches are applied for the tumor diagnostics. These methods include screening blood circulating biomarkers, biopsy, and various imaging techniques. The blood circulating biomarkers hold great potential for the tumor diagnostics. In the current stage, however, it lacks sensitivity and specificity. For example, the PSA test, a standard for prostate cancer diagnostics, has a low correlation with prostate cancer staging. Biopsy is an informative method. However, this method is extremely invasive. The patients have to suffer from the great pain before the tissues are taken out from the body for the analysis. The “golden-standard” the evaluation of angiogenesis is the mean vascular density (MVD), which estimates the blood vessel density by IHC. However, tumor is usually heterogeneous, and MVD only quantify limited area in the collected tissues in the specific location of the tumor. Thus, the MVD of biopsy tissue can be misleading.64, 69 On the other hands, imaging techniques are used to detect angiogenesis. However, most of these techniques are not satisfied with the requirement for the tumor diagnostics. The fluorescence imaging lacks of the tissue penetration. The sensitive imaging techniques such as PET and SPECT are expensive, and the number of PET and SPECT scanner is limited. Besides, PET and SPECT imaging use radioactive isotopes, which could be potentially harmful to the patients.64 
MRI is one of the promising techniques for the cancer diagnostics. Magnetic renascence Imaging (MRI) is an advanced diagnostic technique with high resolution, three dimensional properties. Limited by the low sensitivity, 30% of the MRI tests require the administration of MRI contrast agents to improve the signal-to-noise ratio and obtain tissue-specific images.26 With 7 unpaired protons, high magnetic moment and long election relaxation time, Gd3+ became the best metal ion function as contrast agents. Gd3+-based MRI contrast agents were applied for current clinically available diagnostics for over two decades.
The low sensitivity of MRI contrast agents limits the application of this technique for molecular imaging. To image the biomarkers with limited receptor number, MRI contrast agents must be incorporated with targeting moieties and have high relaxivity. Unfortunately, most of the current contrast agent only has relatively low relaxivity (less than 5 s−1 mM−1 for Gd-DTPA), and the lowest detection limits for these contrast agents are 0.1 mM. Therefore, it is hard to image the expression level of certain receptors when the receptor number is low. On the other hand, to targeting to the receptors, these contrast agents are usually conjugated to antibodies or peptides. Unfortunately, even single chain antibody has a large molecular weight and contrast agents conjugated with antibodies have low penetration to tumor tissue.34 Protein-based contrast agents have more than 10 fold higher relaxivity than that of the current clinically available MRI contrast agents. By conjugating to small peptides or affibody, ProCA1s is able to image biomarkers, such as HER-2 and GRPR.